Dependent Nonhomologous End Joining Research Articles

Targeting of Quiescent and Proliferating CML Stem Cells By DNA Repair Inhibitors

Tyrosine kinase inhibitors (TKIs) such as imatinib, dasatinib and nilotinib revolutionized the treatment of chronic myeloid leukemia in chronic phase (CML-CP). However, it is unlikely that TKIs will "cure" a majority of CML patients due to the presence of TKI-refractory quiescent leukemia stem cells (LSCs) and/or TKI-resistant proliferating LSCs. Moreover, a sub-population of patients does not respond favorably to TKI therapy and/or accumulates TKI-resistant BCR-ABL1 mutants and additional chromosomal aberrations (high risk/poor responders = HR/PR). This process may lead to the disease relapse and/or malignant progression to fatal chronic myeloid leukemia in accelerated phase or blast phase (CML-AP/BP). Therefore new treatment modalities are necessary to improve therapeutic outcome of CML HR/PRs.We found that CML LSCs and LPCs, including quiescent LSCs, from HR/PRs accumulate the highest levels of reactive oxygen species (ROS)-induced DNA double strand breaks (DSBs) in comparison to "good responders" and normal counterparts (Bolton-Gillespie et al., Blood, 2013). DSBs are the most lethal DNA lesions, but CML cells can tolerate them because two major repair mechanisms, homologous recombination (HR) and non-homologous end-joining (NHEJ), are hyper-activated (Slupianek et al., Mol. Cell, 2001; Oncogene, 2005; DNA Repair, 2006; Cancer Res., 2011; Blood, 2011; Nowicki et al., Blood, 2005). It appears that CML cells, and especially these from HR/PRs are "addicted" to DSB repair pathways to survive pro-apoptotic challenge from lethal DSBs.There are critical differences between DSB repair in normal and CML cells due to downregulation of BRCA1 in HR pathway and DNA-Pkcs in NHEJ pathway (Slupianek et al., Cancer Res., 2011; Podszywalow-Bartnicka et al., Cell Cycle, 2014). Therefore, proliferating LSCs/LPCs employ RAD52-dependent alternative HR, in contrast to BRCA1-mediated HRR in normal counterparts. Quiescent LSCs use PARP1-mediated alternative NHEJ instead of DNA-PKcs -dependent NHEJ, which is predominant in normal quiescent HSCs. We hypothesized that simultaneous targeting of PARP1 and RAD52 will trigger "dual synthetic lethality" eradicating quiescent LSCs and proliferating LSCs/LPCs.To test this hypothesis we generated Parp1-/-Rad52-/- double knockout mice, which did not display any detectable problems in hematopoietic system and other organs. Leukemogenesis in SCLtTA/p210BCR-ABL1/Parp1-/-Rad52-/- mice was much prolonged in comparison to SCLtTA/p210BCR-ABL1/Parp1-/-, SCLtTA/p210BCR-ABL1/Rad52-/- and SCLtTA/p210BCR-ABL1 animals; 33% of SCLtTA/p210BCR-ABL1/Parp1-/-Rad52-/- mice did not develop leukemia. Moreover, BCR-ABL1 transfected Parp1-/-Rad52-/- bone marrow cells formed the lowest number of colonies when compared to BCR-ABL1 transfected Parp1-/-, Rad52-/-, and wild-type cells.Next, we tested anti-leukemia effect of a combination of PARP1 inhibitor (FDA approved Lynparza) and RAD52 small molecule inhibitor (RAD52smi) identified by high-throughput screen. Simultaneous administration of RAD52 and PARP1 inhibitors completely eradicated clonogenic activity of TKI-treated CML-CP HR/PRs and CML-AP LSCs/LPCs and almost exhausted quiescent LSCs without affecting normal cells. These inhibitors were also very effective against Ph+ ALL cells.In conclusion "dual synthetic lethality" triggered by simultaneous targeting of PARP1 and RAD52 represents a novel strategy which may eradicate quiescent LSCs and proliferating LSCs/LPCs not only from CML but also from BCR-ABL1 -positive ALL. DisclosuresValent:Novartis: Consultancy, Honoraria, Research Funding; Celgene: Honoraria; Bristol-Myers Squibb: Honoraria; Ariad: Honoraria, Research Funding; Pfizer: Honoraria.

The repair of environmentally relevant DNA double strand breaks caused by high linear energy transfer irradiation – No simple task

High linear energy transfer (LET) ionising radiation (IR) such as radon-derived alpha particles and high mass, high energy (HZE) particles of cosmic radiation are the predominant forms of IR to which humanity is exposed throughout life. High-LET forms of IR are established carcinogens relevant to human cancer, and their potent mutagenicity is believed, in part, to be due to a greater incidence of clustered DNA double strand breaks (DSBs) and associated lesions, as ionization events occur within a more confined genomic space. The repair of such DNA damage is now well-documented to occur with slower kinetics relative to that induced by low-LET IR, and to be more reliant upon homology-directed repair pathways. Underlying these phenomena is the relative inability of non-homologous end-joining (NHEJ) to adequately resolve high-LET IR-induced DSBs. Current findings suggest that the functionality of the DNA-dependent protein kinase (DNA-PK), comprised of the Ku70-Ku80 heterodimer and the DNA-PK catalytic subunit (DNA-PKcs), is particularly perturbed by high-LET IR-induced clustered DSBs, rendering DNA-PK dependent NHEJ less relevant to resolving these lesions. By contrast, the NHEJ-associated DNA processing endonuclease Artemis shows a greater relevance to high-LET IR-induced DSB repair. Here, we will review the cellular response to high-LET irradiation, the implications of the chronic, low-dose modality of this exposure and molecular pathways that respond to high-LET irradiation induced DSBs, with particular emphasis on NHEJ factors.

Systems Modelling of NHEJ Reveals the Importance of Redox Regulation of Ku70/80 in the Dynamics of DNA Damage Foci

The presence of DNA double-stranded breaks in a mammalian cell typically activates the Non-Homologous End Joining (NHEJ) pathway to repair the damage and signal to downstream systems that govern cellular decisions such as apoptosis or senescence. The signalling system also stimulates effects such as the generation of reactive oxygen species (ROS) which in turn feed back into the damage response. Although the overall process of NHEJ is well documented, we know little of the dynamics and how the system operates as a whole. We have developed a computational model which includes DNA Protein Kinase (DNA-PK) dependent NHEJ (D-NHEJ) and back-up NHEJ mechanisms (B-NHEJ) and use it to explain the dynamic response to damage induced by different levels of gamma irradiation in human fibroblasts. Our work suggests that the observed shift from fast to slow repair of DNA damage foci at higher levels of damage cannot be explained solely by inherent stochasticity in the NHEJ system. Instead, our model highlights the importance of Ku oxidation which leads to increased Ku dissociation rates from DNA damage foci and shifts repair in favour of the less efficient B-NHEJ system.

Targeting Abnormal DNA Repair in Therapy-Resistant Breast Cancers

Although hereditary breast cancers have defects in the DNA damage response that result in genomic instability, DNA repair abnormalities in sporadic breast cancers have not been extensively characterized. Recently, we showed that, relative to nontumorigenic breast epithelial MCF10A cells, estrogen receptor-positive (ER+) MCF7 breast cancer cells and progesterone receptor-positive (PR+) MCF7 breast cancer cells have reduced steady-state levels of DNA ligase IV, a component of the major DNA-protein kinase (PK)-dependent nonhomologous end joining (NHEJ) pathway, whereas the steady-state level of DNA ligase IIIα, a component of the highly error-prone alternative NHEJ (ALT NHEJ) pathway, is increased. Here, we show that tamoxifen- and aromatase-resistant derivatives of MCF7 cells and ER(-)/PR(-) cells have even higher steady-state levels of DNA ligase IIIα and increased levels of PARP1, another ALT NHEJ component. This results in increased dependence upon microhomology-mediated ALT NHEJ to repair DNA double-strand breaks (DSB) and the accumulation of chromosomal deletions. Notably, therapy-resistant derivatives of MCF7 cells and ER(-)/PR(-) cells exhibited significantly increased sensitivity to a combination of PARP and DNA ligase III inhibitors that increased the number of DSBs. Biopsies from ER(-)/PR(-) tumors had elevated levels of ALT NHEJ and reduced levels of DNA-PK-dependent NHEJ factors. Thus, our results show that ALT NHEJ is a novel therapeutic target in breast cancers that are resistant to frontline therapies and suggest that changes in NHEJ protein levels may serve as biomarkers to identify tumors that are candidates for this therapeutic approach.

Error-prone nonhomologous end joining repair operates in human pluripotent stem cells during late G2

Genome stability of human embryonic stem cells (hESC) is an important issue because even minor genetic alterations can negatively impact cell functionality and safety. The incorrect repair of DNA double-stranded breaks (DSBs) is the ultimate cause of the formation of chromosomal aberrations. Using G2 radiosensitivity assay, we analyzed chromosomal aberrations in pluripotent stem cells and somatic cells. The chromatid exchange aberration rates in hESCs increased manifold 2 hours after irradiation as compared with their differentiated derivatives, but the frequency of radiation-induced chromatid breaks was similar. The rate of radiation-induced chromatid exchanges in hESCs and differentiated cells exhibited a quadratic dose response, revealing two-hit mechanism of exchange formation suggesting that a non-homologous end joining (NHEJ) repair may contribute to their formation. Inhibition of DNA-PK, a key NHEJ component, by NU7026 resulted in a significant decrease in radiation-induced chromatid exchanges in hESCs but not in somatic cells. In contrast, NU7026 treatment increased the frequency of radiation-induced breaks to a similar extent in pluripotent and somatic cells. Thus, DNA-PK dependent NHEJ efficiently participates in the elimination of radiation-induced chromatid breaks during the late G2 in both cell types and DNA-PK activity leads to a high level of misrejoining specifically in pluripotent cells.

DNA double strand break repair inhibition as a cause of heat radiosensitization: Re-evaluation considering backup pathways of NHEJ

Heat shock is one of the most effective radiosensitizers known. As a result, combination of heat with ionizing radiation (IR) is considered a promising strategy in the management of human cancer. The mechanism of heat radiosensitization has been the subject of extensive work but a unifying mechanistic model is presently lacking. To understand the cause of excessive death in irradiated cells after heat exposure, it is necessary to characterize the lesion(s) underlying the effect and to determine which of the pathways processing this lesion are affected by heat. Since DNA double strand breaks (DSBs) are the main cause for IR-induced cell death, inhibition of DSB processing has long been considered a major candidate for heat radiosensitization. However, effective radiosensitization of mutants with defects in homologous recombination repair (HRR), or in DNA-PK dependent non-homologous end joining (D-NHEJ), the two primary pathways of DSB repair, has led to the formulation of models excluding DSBs as a cause for this phenomenon and attributing heat radiosensitization to inhibition of base damage processing. Since direct evidence for a major role of base damage in heat radiosensitization, or in IR-induced killing for that matter, is scarce and new insights in DSB repair allow alternative interpretations of existing data with repair mutants, we attempt here a re-evaluation of the role of DSBs and their repair in heat radiosensitization. First, we reanalyse data obtained with various DSB repair mutants on first principles and in the light of the recent recognition that alternative pathways of NHEJ, operating as backup (B-NHEJ), substantially contribute to DSB repair and thus probably also to heat radiosensitization. Second, we review aspects of combined action of heat and radiation, such as modulation in the cell-cycle-dependent variation in radiosensitivity to killing, as well as heat radiosensitization as a function of LET, and examine whether the observed effects are compatible with DSB repair inhibition. We conclude with a model reclaiming a central role for DSBs in heat radiosensitization.

Up-Regulated WRN and DNA Ligase IIIα Are Involved in Alternative NHEJ Repair Pathway of DNA Double Strand Breaks (DSB) in Chronic Myeloid Leukemia (CML).

The oncogenic BCR-ABL in CML produces increased reactive oxygen species (ROS) leading to DSB and aberrant repair. We have previously shown that CML cells demonstrate an increased frequency of errors of non homologous end-joining (NHEJ). DSB are repaired by two major pathways, homologous recombination (HR) and NHEJ, the dominant pathway in eukaryotic cells, also known as DNA-PK dependent NHEJ (D-NHEJ). Recent reports have identified alternative or “back-up” NHEJ pathways (B-NHEJ) that are highly error-prone, and may explain the altered DSB repair reported in CML. To determine the mechanism for the aberrant NHEJ repair in CML, we examined steady state levels of D-NHEJ proteins, including Ku70/86, DNA-PKcs, Artemis and DNA Ligase IV/XRCC4 in four different BCR-ABL positive CML cell lines compared with three lymphoblastoid cell lines established from normal individuals and one BCR-ABL negative CML cell line. We find that two key components of D-NHEJ, Artemis (4–7 fold) and DNA Ligase IV (2–3 fold) are down-regulated, compared with controls. These data suggest that D-NHEJ repair is compromised in CML. To determine whether alternative NHEJ repair plays a role in the aberrant repair of DSB in CML cells, we next examined expression levels of DNA Ligase IIIα/XRCC1, PARP and other proteins known to be associated with NHEJ repair, such as the protein found to be deleted in Werner's syndrome, WRN. We find that WRN and DNA Ligase IIIα are increased (3–6 fold) in BCR-ABL-positive CML compared with control cell lines. Importantly, DNA Ligase IIIα/XRCC1 forms a complex with WRN, suggesting that it may be a new member of the alternative repair pathway. To confirm that up-regulation of DNA Ligase IIIα and WRN are elicited by BCR-ABL, we examined the levels of these proteins in primary samples (N=4) from patients with different levels of BCR-ABL, following treatment with the tyrosine kinase inhibitor Gleevec. WRN and DNA Ligase IIIα are down regulated in patient samples where BCR-ABL levels are significantly decreased. Furthermore, we confirmed that these up-regulated proteins are involved in DSB repair in CML cells because they co-localize to induced DSB in BCR-ABL-positive cell lines stably transfected with DSB-containing DRneo plasmid, using fluorescence in situ hybridization (FISH) co-immunostaining. Importantly we show that siRNA down-regulation of WRN and DNA Ligase IIIα leads to elevated levels of unrepaired DSB and a decreased frequency of DSB repair efficiency in CML cells. In addition siRNA down-regulation of WRN leads to large deletions at the site of repair, while siRNA down-regulation of DNA Ligase IIIα results in an increased frequency of misrepair. Finally, we determined whether “correction” of main NHEJ pathway proteins in CML can lead to a decrease in the frequency of errors of end-joining repair. Over-expression of Artemis using pcDNA constructs in CML cells leads to more correct end-joining, compared with vector transfected controls. We conclude that down-regulation of Artemis and DNA Ligase IV leads to compensatory up-regulation of alternative repair pathways in BCR-ABL-positive CML cells, and suggest a role for a new protein complex in CML, in protecting and joining DNA ends, thus ensuring the survival of CML cells. Inhibition of alternative NHEJ repair may be explored in combination with other agents as a therapeutic strategy in CML.